Cancer is a progressive disease culminating in acquisition of metastatic potential by a subset of evolving tumor cells. Generation of an adequate blood supply in tumors by production of new blood vessels, angiogenesis, is a defining element in this process. Although extensively investigated, the precise molecular events underlying tumor development, cancer progression and angiogenesis remain unclear. Subtraction hybridization identified a novel genetic element, progression elevated gene-3 (PEG-3), whose expression directly correlates with cancer progression and acquisition of oncogenic potential by transformed rodent cells. As demonstrated herein, forced expression of PEG-3 in tumorigenic rodent cells, and in human cancer cells, increases their oncogenic potential in nude mice as reflected by a shorter tumor latency time and the production of larger tumors with increased vascularization. Moreover, inhibiting endogenous PEG-3 expression in progressed rodent cancer cells by stable expression of an antisense expression vector extinguishes the progressed cancer phenotype. Cancer aggressiveness of PEG-3 expressing rodent cells correlates directly with increased RNA transcription, elevated mRNA levels and augmented secretion of vascular endothelial growth factor (VEGF). Furthermore, transient ectopic expression of PEG-3 transcriptionally activates PEG-3 in transformed rodent and human cancer cells. Taken together these data demonstrate that PEG-3 is a positive regulator of cancer aggressiveness, a process regulated by augmented VEGF production. These studies also support an association between expression of a single non-transforming cancer progression inducing gene, PEG-3, and the processes of cancer aggressiveness and angiogenesis.
Genetic changes implicated in cancer development and progression include oncogene activation and tumor suppressor gene inactivation (1–4). Recent studies suggest an additional component to this paradigm, involving genes that are associated with and may directly mediate (progression elevated genes, (PEGen)) or suppress (progression suppressed genes, (PSGen)) cancer aggressiveness and tumor progression (3,4). One PEGen, PEG-3, was identified as a gene displaying elevated expression as a consequence of cancer progression and DNA damage in rodent tumor cells (3). A fundamental question in cancer biology is the mechanism by which these diverse genetic elements interact in mediating tumor development and progression.
An important event in controlling the growth of both primary and metastic tumors is angiogenesis (5–9). Without neovascularization (formation of new blood vessels), tumors usually do not grow beyond a few mm3 in size (5–7). The formation of new tumor-associated neovascularization is responsible for the increased perfusion of nutrients and oxygen into the tumor mass and the removal of waste products. This process also facilitates entry of tumor cells into the circulatory system, a prerequisite for metastasis. Consistent with this finding, a high degree of tumor vascularization directly correlates with an increase in a tumor's malignant phenotype and inversely correlates with patient survival (10–12). Production of new blood vessels by the developing tumor and distant metastases results from the elaboration if large quantities of angiogenic molecules by both the tumor and host cells (5–9). The balance between positive and negative regulators of this process (8,9) controls the degree of angiogenesis. These observations emphasize that any genetic modification in a cancer cell that culminates in expansion of tumor growth and metastasis will be inexorably linked to angiogenesis.
Transformation of early passage rat embryo cells by adenovirus type 5 (Ad5) is a progressive process in which morphologically transformed cells temporally acquire new and exhibit further elaboration of existing transformation-related properties (1,13,14). Isolating cells after growth in agar, co-expressing additional oncogenes or reisolating transformed cells after tumor formation in nude mice (13–15) can accelerate this process. Substraction hybridization of a cDNA library generated from a mutant Ad5 (H5ts125)-transformed rat embryo cell clone that forms small, slow-growing and compact tumors, E11 (1,13,14), from a cDNA library produced from a highly aggressive tumorigenic nude mouse tumor-derived E11 clone, E11-MT (2,14), resulted in the identification and cloning of PEG-3 (3). Elevated PEG-3 expression occurs in progressed H5ts125-transformed clones and in normal rat embryo fibroblast (CREF) (16) cells displaying a tumorigenic phenotype as a result of expression of diverse acting oncogenes, including Ha-ras, V-src, human papilloma virus type-18 transforming genes and a specific mutant of Ad5 (H5 hr1) (3). When PEG-3 is ectopically expressed in E11 cells, anchorage-independence, an in vitro marker of progression in this model system, is increased (3). These results indicate that PEG-3 can directly contribute to expression of the in vitro transformed phenotype in H5ts125-transformed rat embryo cells.
A number of questions remain concerning the potential role of PEG-3 in regulating the cancer phenotype. These include the biological consequence of elevating PEG-3 expression in normal cells and in vivo outcome of modifying PEG-3 expression in cancer cells. In the present study we demonstrate that PEG-3 lacks classical oncogenic potential, but overexpression of this gene in rodent or human tumor cells results in aggressive tumorigenic properties in athymic nude mice. The phenotypic changes induced by over expression of PEG-3 correlate with an increase in VEGF production. These findings provide a potential mechanistic framework by which PEG-3 enhances the in vivo cancer phenotype to tumor cells.